At d7, TRIF deficiency reduced transcription factors and TRIF-dependent cytokines. Through the modulation of the TGF-β-signaling pathway and anti-fibrotic microRNAs, TRIF was involved in the development of interstitial fibrosis. The absence of TRIF was associated with a decreased expression of proapoptotic proteins. In echocardiography and working heart analyses, TRIF deficiency slowed left-ventricular wall thickening, myocardial hypertrophy, and reduces the ejection fraction. In summary, TRIF is an important adapter protein for the release of inflammatory cytokines and the accumulation of inflammatory cells in the early stage of maladaptive cardiac remodeling. TRIF is involved in the development of cardiac fibrosis by modulating inflammatory and fibrotic signal transduction pathways.Pressure-overload-induced cardiac hypertrophy represents one cause of the development of heart failure. The aim of this study is to characterize the influence of the TIR-domain-containing adapter-inducing interferon-β (TRIF) during afterload-induced myocardial remodeling. After trans-aortic constriction (TAC), cardiac pressure overload leads to an early increase in MyD88- (Myeloid differentiation primary response gene 88) and TRIF-dependent cytokines. The maximum cytokine expression appeared within the first week and decreased to its control level within five weeks. While cardiomyocyte hypertrophy was comparable, the myocardial accumulation of the inflammatory cells was lower in TRIF Increased afterload, as imposed by arterial hypertension or aortic stenosis, results in pathological myocardial hypertrophy, which has to be differentiated from physiological hypertrophy in pregnancy or exercise ,2 and isDuring maladaptive myocardial remodeling, the continued loss of cardiomyocytes and the replacement by interstitial fibrosis  leads toCardiac remodeling is triggered by cardiac injuries, cytokine secretions, and enhanced autonomic activity . VariousWe hypothesized that TLR3/4-TRIF inflammatory signaling is involved in cardiac remodeling processes in afterload-induced myocardial hypertrophy. In this study, we determined the time of maximal cytokine expression in TRIF knockout and wild-type mice after trans-aortic constriction (TAC). Furthermore, we focused on inflammatory cell infiltration and fibrosis at the time-point of maximal cytokine expression (day 7).The study was approved by the animal Ethics Committee of Saarland University des and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .Age- and sex-matched TRIF-deficient mice (Ticam1Lps2/J)   with the RMV-707B transducer .The mice were anesthetized with 1.5% isoflurane and imaged using the High-Resolution in vivo Micro-Imaging System Vevo®770 Version 2.3.0  Software. The following functional parameters were calculated from the M-mode measurements: Left ventricular mass (LV mass [g]) and left ventricular end-diastolic volume (LVED vol [μL]), as well as the left ventricular shortening fraction ; see In parasternal longitudinal axis projection after the acquisition of the apex and the aortic root in one line, two-dimensional (B-mode) images were taken. At the end diastole and end systole, apical to the papillary muscle, one-dimensional M-mode measurements were taken in the same setting. The left ventricular cavity sizes and wall thicknesses were observed and averaged in this projection for three heart cycles. The interventricular septum , the left ventricular internal diameter at end-diastole (LVIDd/LVEDD [mm]) and end-systole (LVIDs/LVESD [mm]) and the left ventricular posterior wall  were recorded. In the short-axis projection, the standard measurement of the left ventricular anterior wall  was also performed at three heart cycles. The data were collected and analyzed with the VisualSonics Vevo®-Rompun® anesthetic mixture . Immediately after death, the heart was excised and transferred into an ice-cold cardioplegic solution. After the removal of the pericardium, lung remains, and trachea, the aorta was cannulated with an 18 g metal cannula and perfused in the Working Heart apparatus with Krebs–Henseleit buffer and a perfusion pressure of 60 mmHG. After the sufficient perfusion of the heart in the Langendorff mode, a 16 g steel cannula was positioned in the left atrium via the pulmonary vein. As part of the Working Heart preparation, the cannula was then connected to a column filled with Krebs–Henseleit buffer and heated to 38 °C . Two platinum electrodes embedded in polyester resin were attached to the right atrium, stimulating the heart rate to approximately 400 beats per minute (bpm).The studies on the isolated hearts were carried out as previously described . BrieflyThe left ventricular systolic and diastolic functions were measured using a pressure–volume catheter . The catheter was inserted into the left ventricle with a 22 ¼ g needle after puncturing the apex. The end-diastolic pressure–volume ratio (ESPVR), and the associated gradient of EES (end-systolic ventricular elasticity), were assessed by increasing the afterload from 60 mmHg to 100 mmHg. The preload was left constant at 10 mmHG and the heart rate at 400 bpm. Finally, the wall thickness was determined by adding 5 µL of hypertonic saline solution (5%) via the cannula into the left atrium (transient change in the conductivity of the Krebs–Henseleit buffer in the left ventricular cavity).v/v) fetal calf serum, gentamicin (0.08 mg/mL), and penicillin (100 IU/mL). The cells were used for the experiments when sub-confluent in passage 3. HUVECS were grown in Endothelial-Cell-Growth medium  and used in passages 3 to 6.Neonatal rat cardiomyocytes and cardiac fibroblasts were isolated from 5-day-old neonatal Sprague Dawley rat hearts (Charles River) of mixed sex, as described . BrieflyNeonatal rat cardiomyocytes, neonatal cardiac fibroblasts, or HUVECs were transfected with 15 nM TRIF-siRNA or negative siRNA using the HiPerfect Transfection Reagent . The cells were harvested for 48 h and then isolated for protein expression and cell proliferation tests.The paraffin-embedded LV heart tissues of the transverse cross-sections (3 µm) were stained with hematoxylin/eosin for the cardiomyocyte area. Several photographs were taken to cover the whole cross-section of the embedded heart. The pictures were magnified electronically, and the cross-sectional area of 100 cells, with nuclei approximately in the center of the area, was microscopically determined with the LUCIA G© Software  at 40× magnification.Picrosirius-red was performed for the analysis of fibrosis. The collagen content of the cross-section was determined by marking the red areas via LUCIA G© Software . The collage content [%] = area of collagen/area of the cross-section.The hydroxyproline content was assessed using QuickZyme Hydroxyproline Assays . After 100 mg of tissue was hydrolyzed, hydroxyproline was determined via the ELISA technique. The content was calculated via a standard curve.Immunofluorescence staining was performed for the detection of cardiomyocytes , macrophages , and T-lymphocytes  on paraffin-embedded LV heart tissues of the transverse cross-sections (3 µm). After heat-mediated antigen retrieval with citraconic anhydride solution, incubation with the first antibody was performed by overnight incubation at 4 °C, additional at 37 °C for 1 h, and subsequently with the appropriate secondary antibody at 37 °C for 1 h. In 15 areas at 100× magnification, positive cells were counted against all of the DAPI-positive cardiomyocytes.® Peroxidase In Situ OligoLigation (ISOL) Apoptosis Detection Kit, Millipore. The ratio of apoptotic and vital cells in 10 areas was determined for quantification.The apoptotic cells were stained on slides (cross-sections of the heart) with the ApopTagThe RNA from the frozen heart tissue was extracted using the peqGOLD RNAPure solution according to the manufacturer’s instructions (Peqlab Biotechnologie). RNAs from the spleen-derived macrophages and from the cultured cells were isolated with an absolute RNA Microprep Kit . cDNA synthesis was performed with a high-capacity cDNA reverse-transcription kit . The quantitative gene expression was measured using the TaqMan system with KAPA SYBR FAST Universal Sybr Green (Peqlab Biotechnologie) and custom-made primers. The heart tissue and macrophages were examined for the expression of different cytokines See  and appr® MicroRNA Assay (Life Technologies).The detection of microRNA miR29 and miR146a was performed from isolated RNA using the TaqManThe protein extracts from the frozen heart tissues and the si-transfected cells were separated by a SDS PAGE (12%) and transferred to nitrocellulose membranes , followed by the blocking of the non-specific protein with 5% dried milk for 1 h. The blocked membranes were incubated overnight at 4 °C with the primary antibody against IRF3 , TGFβ , TGFβ-RI, TGFβ-RII, p53, p16, Bax and BCL, and GAPDH . The detection was performed using the suitable horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature in 5% dried milk. The plots were quantified by densitometry and normalized against GAPDH.Neonatal rat cardiomyocytes, neonatal cardiac fibroblasts, or HUVECs were seeded in 96-well cell-culture plates, and si-transfection was performed. After 48 h, the cells were used in the Cell Proliferation ELISA, BrdU , according to the manufacturer’s instructions.p < 0.05 were considered significant.The results were expressed as mean + SEM. A statistical comparison between the groups was evaluated using one-way ANOVA and additional Bonferroni’s multi-comparison test. Values of The maximum (five- to nine-fold) cytokine mRNA expression of the cytokines  was found at 3 and 7 days following TAC in the left ventricular (LV) tissue a.p = n.s.; TRIF−/− Sham = 0.71 ± 0.24; TRIF−/− TAC = 0.33 ± 0.18; p = n.s.)   b. The di < 0.01) c.−/− mice. However, following TAC, the gene expression was clearly attenuated in the TRIF−/− mice  and ventricle occurred later at day 35. Similarly, the reduction in the ejection fraction (EF [%]) was delayed in the TRIF−/− mice.In the TRIF−/−mice. The functionality of the hearts was examined on day 35 and day 70 with the Langendorff perfusion method in the working heart mode. A moderate reduction in the ejection fraction  and thtput CO, c). Thesetput CO, .p < 0.01; TRIF−/− Sham = 5.63 ± 0.30; TRIF−/− TAC = 6.46 ± 0.33; p < 0.01) and 50% on day 35 . The increase was similar in both groups  and 60% at d35   b.2 was 12-fold higher in the WT group but only eight-fold higher in the TRIF−/− group after TAC compared to the Sham-operated animals . On day 35, the number of inflammatory T-cells per mm2 was lowered and the difference between the Sham- and TAC-operated animals of each mouse strain was similar  . On day 35, the amount in the heart tissue was nearly identical in the TAC- and Sham-operated mice  a. The map = n.s. b.p < 0.05), while in the TRIF−/− mice, the collagen content was attenuated : The difference between the two groups was considerably pronounced on day 35. The TRIF−/− mice showed less collagen content in the heart tissue than the WT mice    and at d35    a. Simila < 0.01) b. Concer < 0.01) a. In add = n.s.) b.p < 0.05), neonatal rat fibroblasts , and neonatal rat cardiomyocytes , showed a remarkably lower TGFβ protein expression in all cell types . miR-146, which represses SMAD4, showed a higher expression level in the Sham-operated TRIF −/− mice than in WT. After TAC, the miR-146 expression increased in both groups, but the relative expression of miR-146 remained significantly higher in the TRIF−/−group . . .p < 0.05), while in the TRIF−/− mice, apoptosis was more moderate . Either the p16 protein expression increased only in the control mice after TAC . The basic level of the bax/bcl ratio was higher in the C57BL/6 compared to the TRIF−/− mice. After TAC, the bax/bcl ratio increased in the WT but not in the TRIF−/− mice  (p = n.s.) and p16  was decreased after TRIF knockdown. According to that investigation the bax/bcl ratio  was attenuated in the TRIF si-transfected cardiomyocytes  b. In vitmyocytes .Inflammation plays an important role in the adaptive and maladaptive remodeling of the heart. Left-ventricular overload results in myocardial inflammation with the myocardial overexpression of pro-inflammatory cytokines and leukocyte infiltration into the myocardium. Various observational studies in animals and patients have indicated a relationship between the presence of pro-inflammatory markers and pressure overload-induced hypertrophy and fibrosis ,8,9. In −/− mice compared with control at an early (d7) and late stage (d35) following TAC.In our study, while MyD88 knockdown yields reduced cardiac hypertrophy after MI and TAC ,25, TRIF−/− mice in the early stage. These cell types are known as common sources of inflammatory cytokines. The reduced number of these cells could be the cause of the decreased expression of inflammatory cytokines in the heart tissue of the TRIF−/− mice after TAC. During further remodeling, changes in cellular accumulation seemed to vanish, most likely because cellular infiltration is an early process. Because of the role of T-lymphocytes and macrophages in the remodeling processes, other immunomodulatory cell (sub-)types were not analyzed, which is a limitation of the study.However, TRIF knockout led to the downregulation of IRF3 and reduced the downstream gene expression of CXCL10, CXCL11, and CCL5 in LV tissue and in the LPS-stimulated spleen-derived monocytes. Interestingly, differential gene expression was more pronounced locally in heart tissue, possibly because here, following TAC, TRIF inflammatory signaling is strongly switched on. We observed a decrease in T-Lymphocytes and macrophages in the heart tissue of the TAC-operated TRIF−/− mice.Richards et al.  showed t−/− mice comes along with decreased TGFβ protein expression. Through its pleiotropic effects, TGFβ is a key mediator in the transition from inflammation to fibrosis. It exerts effects on all cell types involved in cardiac injury, repair, and remodeling [−/− mice after TAC, which led to less signal transduction in the TGFβ induced pathway. In our in vitro experiments, following TRIF knockdown, the protein expression of TGFβ was downregulated in macrophages, fibroblasts, and cardiomyocytes, suggesting that these cell types are equally involved in TGFβ-mediated fibrosis.Reduced LV fibrosis in the TRIFmodeling . TGFβ re−/− mice, miR-146a was upregulated. Our results emphasized miR-146a as part of the TRIF-regulated TGF-beta signaling pathway.Several studies demonstrated the functional role of microRNAs in cardiac remodeling, particularly during cardiac fibrosis ,35,36,37−/− mice showed similar protein-expression patterns as cell culture experiments. Chen et al. [Cardiac remodeling goes along with altered apoptosis and proliferation. Previous studies on MI showed that TLR3-TRIF signaling contributes to ischemic myocardial injury, most likely by mediating cardiac apoptosis, and plays a lesser role in myocardial inflammation during I/R . We demon et al.  indicateIn summary, the Toll-like adapter TRIF influences afterload-induced myocardial remodeling at several molecular and cellular sites. In the early stage of afterload-induced remodeling, TRIF is involved in intracellular inflammatory signaling by supporting inflammatory cell  -infiltration and modifying inflammatory chemokine gene expression. TRIF signaling enhances apoptosis, attenuates the proliferation of cardiomyocytes, and stimulates the proliferation of fibroblasts. TRIF is involved in the TGFβ-dependent fibrotic pathway. The downregulation of TRIF leads to profoundly reduced fibrosis via TGFβ signaling and the increased expression of anti-fibrotic microRNAs . In summ


Further, to achieve an effective photothermal effect, H-SWNTs were selected as nanofillers to reduce energy loss while increasing light-absorption efficiency. Thereafter, near-infrared rays of 818 nm were directly irradiated onto the nanocomposite film to induce photothermal properties arising from the local surface plasmon resonance effect on the CNT surface. A self-heating shape memory composite material that rapidly heated to 270 °C within 1 min was developed, even when only 3 wt.% of H-SWNTs were added. The results of this study can be used to guide the development of heat-generating coating materials and de-icing materials for the wing and body structures of automobiles or airplanes, depending on the molding method.In this study, high-crystallinity single walled carbon nanotubes (H-SWNTs) were prepared by high-temperature thermal annealing at 1800 °C and a self-heating shape memory polyurethane nanocomposite with excellent self-heating characteristics was developed within a few seconds by irradiation with near-infrared rays. With a simple method (heat treatment), impurities at the surface of H-SWNTs were removed and at the same time the amorphous structure converted into a crystalline structure, improving crystallinity. Therefore, high conductivity  and interfacial affinity with PU were increased, resulting in improved mechanical, thermal and electric properties. The electrical conductivity of neat polyurethane was enhanced from ~10 Polyurethane (PU) is a representative engineering plastic with excellent mechanical properties, such as good heat and wear resistance, a high elongation rate, and excellent workability. It has been widely applied in adhesives, coatings, rubber, and biomaterials with good biocompatibility, high chemical resistance, and excellent processability ,2,3,4,5.There are many applications of carbon nanotube (CNT)-based nanocomposites, owing to their high aspect ratio, good charge transport properties, and high electrical and thermal properties ,12. In pVarious surface treatment methods have been developed to alter the thermal and electrical properties of CNTs. For example, high-temperature heat treatment and doping of hetero-elements can allow the control of CNT transport characteristics, which is effective for improving their conductivity by controlling their energy bandgaps; this method can be applied to develop new nanocomposites ,20,21.We previously reported a photothermal effect using near-infrared radiation (NIR) after preparing a PU nanocomposite using boron-doped CNTs . HoweverThe photothermal effect is defined as a physical phenomenon in which, when a material is irradiated with light energy, the irradiated light energy is absorbed and then released as heat according to the properties and characteristics of the material. To use the photothermal effect in a self-heating shape memory composite material, the selection of nanofillers that increase the light absorption efficiency while decreasing the fluorescence quantum yield and controlling the interfacial properties with the polymer matrix should be considered together.CNTs were selected as representative candidates. CNTs can convert absorbed light into heat with strong absorbance over a wider wavelength range than that of metal nanoparticles. The photothermal effect of CNTs depends on their dispersion, type, and morphology on the substrate. These properties have been applied in various fields such as nano-thermal derivatives, photothermal therapy, imaging, actuators, and sensors ,29,30,31In this study, SWCNT nanofillers annealed at high temperatures were combined with PU to produce nanocomposites. In order to overcome the disadvantages of CNT modification through doping method, highly crystalline CNTs were prepared through high-temperature heat treatment. The method through high heat treatment removes impurities on the surface and has high crystallinity through crystallization of an amorphous material, so that CNTs having excellent electrical and mechanical properties can be obtained. The photothermal properties of the nanocomposites were evaluated by irradiating the prepared PU nanocomposite films with NIR light at 808 nm. The characteristics of the filler, content, and degree of dispersion, as well as the mechanical, thermal, and electrical properties of the nanocomposite material were evaluated.Single-walled nanotubes  with an ideal diameter (~1.4–1.7 nm), length (5–20 μm), tap density (0.02 g/cc) and high purity (>95 wt.%) were synthesized by arc discharge process. The highly thermally annealed SWCNTs (H-SWNTs) were obtained by annealing at 1800 °C for 30 min in a graphite furnace operating in an argon atmosphere. The H-SWNTs were then kept in a desiccator for future use.Poly(tetramethylene glycol) , 4,4′-methylene bis(phenylisocyanate) , and 1,4-butandiol  were used for the synthesis of the shape memory PU block copolymer (SHPU) . In a 50The homogeneously dispersed H-SWNT suspension was prepared by adding nanotubes to NMP and then sonicating for 1 h . The synthesized SHPU solution (re-dissolved in NMP) was poured into the nanotube suspension and mixed using a planetary centrifugal mixer . Finally, we prepared a composite film by casting the H-SWNTs/SHPU suspension on chalets and drying at 50 °C for 3 h and at 110 °C for 12 h in vacuum to remove the residual solvent. The resulting samples were obtained as films with thicknesses of 100–120 µm. SHPU nanocomposites with H-SWNT loadings of 1, 3, and 5 wt.% were fabricated, and their specific compositions are summarized in Raman spectroscopy  was used to monitor the changes in the optical properties of the CNTs before and after high-temperature thermal annealing. Wide-angle X-ray diffraction  analysis was performed to identify the structures of neat PU, P-SW@PU, H-HW@PU composites. X-ray photoelectron spectroscopy  was performed using a Mg Kα X-ray source with a 10-mA emission current and a 10 kV accelerating voltage. The thermal stabilities of the nanocomposites were examined using thermogravimetric analysis (TGA)  in the temperature range from room temperature to 900 °C at a rate of 10 °C/min in nitrogen.2. The NIR laser spot size of 0.8–0.5 cm2 was used.Fourier-transform infrared (FT-IR) spectroscopic measurements were performed to identify the structure of the SHPU and H-SWNTs using an IR Prestige-21 (Shimadzu) equipped with an attenuated total reflectance accessory. The cross-sectional morphology of the SHPU nanocomposites was examined by field-emission scanning electron microscopy  using Pt-coated samples. The mechanical properties were evaluated at room temperature according to the ASTM D638 test method using a universal testing machine  with dog-bone-type dumbbell specimens. The dimensions of the specimens were 30 (length) × 5 (width) × 5 (narrow portion length) × 2 (narrow portion width) × 0.20 (thickness) mm. The measurement conditions were as follows: 10 mm gauge length; 20 mm/min crosshead speed; and 100 N load cell. At least five samples were tested, and the average was used. An NIR laser was used to measure the heat-generation characteristics of the nanocomposite materials. Photothermal measurements of the pristine SWNTs and the H-SWNT-incorporated PU nanocomposite were performed using the NIR laser  and the laser power density of 1.5 W/cm−1, it can be seen that the tube diameter was approximately 1.25 nm (deVRBM = 248/d). However, the change in the diameter of the tube owing to heat treatment could not be confirmed. The G-band at 1591 cm−1 and the D-band at 1347 cm−1 resulting from the E2g mode were observed [D/IG (0.003 for H-SWNTs and 0.02 for P-SWNTs), it is clear that the graphite structure and crystalline structure of the heat-treated SWCNT (H-SWNTs) were more developed than those of the P-SWNTs [observed ,33, and  P-SWNTs ,36. To oAs shown in In addition, 2 (284.7 eV), sp3 (285.2 eV), and C=O (286.1 eV), which are carbon-to-carbon bonds in graphite, change significantly even after heat treatment due to the amorphous carbon the outer surface of the tube [2 24.7 eV, s−1 and 1730 cm−1, respectively. In addition, in the case of the nanocomposites (P-SW@PU and H-SW@PU) to which fillers were added before and after heat treatment, the OH functional group at 3500 cm−1 decreased gradually, and the asymmetric C–H stretching bond at 2925 cm−1 and 2845 cm−1 was reduced. However, because the synthesized PU nanocomposite was formed by simple physical mixing with CNTs, no significant changes in the peaks could confirm the reaction between the PU backbone and nanofillers.H-SWNTs with crystallinity developed through high-temperature heat treatment were added to PU to prepare a nanocomposite material, and FT-IR was used to observe changes in the structural properties. In Tensile testing was used to evaluate the mechanical properties of the PU nanocomposites containing nanofillers, and the elastic modulus, breaking strength, and elongation at break were monitored as the filler content was increased to 1, 3, and 5 wt.% based on the weight of PU as shown –11 S/cm) of the neat PU was consistent with the values reported reference [−8 to 1.07 × 10−6 and 4.66 × 10−6 S/cm, respectively. In addition, the thermal conductivity increased by up to 60% from 0.21 to 0.265 and 0.338 W/m·K, respectively. Such a result can be seen as an effect of an increase in the crystallinity of the CNTs, as well as an increase in the filler content.eference . As the In 2) to evaluate the self-heating properties of the developed shape memory PU nanocomposite. In the case of PU, it can be seen that no temperature change occurs on the surface regardless of the presence of laser irradiation. However, the H-SWNT-containing PU nanocomposite was rapidly heated to 270 °C within 1 min. In particular, as the filler content increased, the maximum exothermic temperature tended to increase, but considering that the filler content showed a similar trend at 3 and 5 wt.%, the filler content required for maximum exothermic performance was judged to be around 3 wt.%.In this study, a self-heating nanocomposite material with excellent mechanical, thermal, and electrical properties was manufactured with polyurethane (PU) and the high-temperature heat treated SWCNTs. The high heat treatment was given at 1800 °C to remove the amorphous material from the surface of CNTs and develop more crystalline SWCNTs. First, SWCNTs through high-temperature heat treatment (H-SWNTs) were able to control the structure of CNTs by removing impurities from the surface of CNTs and changing amorphous materials into crystalline structures. Second, when high crystalline H-SWNTs were incorporated instead of P-SWNTs with PU, it shows higher mechanical properties than P-SWNTs. Subsequently, the characteristics of the nanocomposite materials, which were capable of heating from room temperature to 270 °C within 1 min, were investigated by irradiating with NIR light of 818 nm. As a result, using only heat treatment, it was possible to improve the surface characteristics of the nanofiller and develop a material with excellent heat-generating performance, electrical conductivity and mechanical properties.In the future, this self-heating nanocomposite could be used in all industries, from home heaters to large-area heaters for e-mobility, functional windows for construction, showcase heaters, and heater modules in the printing industry.


Increasing evidence has shown that elevated Thyroid stimulating hormone (TSH) levels are positively correlated with atherosclerosis (ATH) in patients with subclinical hypothyroidism (SCH). Some researchers found that the dysfunction of Endothelial Cells (ECs) in SCH plays an important role in the pathogenesis of ATH in SCH, but the association remains controversial.To determine the expression profiles of serum microRNAs critical to the function of Endothelial cells (ECs) may help reanalyze the possible mechanism underlying ATH in SCH and the association between ATH and SCH.We used qRT-PCR to perform microRNA profiling and analysis in normal control subjects (NC), patients with SCH alone (SCH), patients with SCH and ATH (SCH+ATH), and patients with ATH without SCH (ATH).Both miR-221-3p and miR-222-3p showed a decreasing expression trend between the SCH and SCH+ATH groups. In addition, miR-126-3p and miR-150-5p showed a stepwise decrease from the NC to SCH groups and then to the SCH+ATH or ATH group. miR-21-5p was unregulated in the SCH, SCH+ATH, and ATH groups. Furthermore, elevated levels of miR-21-5p in SCH+ATH group were higher than SCH and ATH group. No differences were found in the levels of miR-150, miR-126, miR-221 and miR-222 between the ATH and the SCH+ATH subjects.miR-21-5p may be involved in the atherosclerosis process in patients with SCH (SCH and SCH+ATH groups). miR-150-5p may be sensitive risk markers for predicting endothelial dysfunction in patients with ATH (ATH and SCH+ATH groups). Subclinical hypothyroidism (SCH) is an endocrine and metabolic disorder that is characterized by elevated serum thyroid-stimulating hormone (TSH) above the upper limit of reference with normal serum free thyroid hormones FT4 and FT3) . Recentl and FT3 MicroRNAs (miRNAs) are a class of endogenous, small noncoding RNAs that are 21~23 nucleotides in length . They arvia vascular endothelial growth factor (VEGF) signaling . Blood pressure was measured in the right arm using a standard mercury sphygmomanometer with an appropriately sized cuff while participants were in a seated position. Blood was drawn with minimal trauma from an antecubital vein in the morning after a 12 h overnight fast and then stored at -80°C in aliquots, partly for blood biochemistry analysis and partly for miRNA extraction. Serum TSH, FT3, FT4, nitric oxide (NO), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL-c) and high-density lipoprotein (HDL-c) were measured in accordance with the kit instructions. To reduce the interassay differences, all measurements were performed at the same clinical laboratory of the Second Affiliated Hospital of Shandong First Medical University.According to our previous study , all bas(CIMT) examination of the bilateral carotid arteries using a color ultrasonic diagnostic (Toshiba Aplio 500 Ultrasound Scanner). Subjects rested in the supine position with the head titled backwards while the study was performed in a temperature-controlled room (25 ℃). The bilateral carotid arteries, internal carotid artery (ICA), common carotid artery (CCA) and carotid bulb (sinus) were longitudinally scanned at different angles, and the thickest part of the vessel wall and plaques along the artery wall were explored. CIMT is defined as the visible distance between the haemal junction and the foreign membrane junction. Three measurements were performed near the thickest point of the IMT and at two adjacent points (1 cm upstream and 1 cm downstream). The mean values of the three measurements are represented as the IMT. The maximum values of the CCA, ICA and sinus of carotid artery were regarded as the maximum (max) IMT. The mean intima thickness at the six left and right points was the mean carotid intima thickness  is suitable for the normalization of circulating miRNA preparation. It was introduced as a spike-in control to monitor technical variations in RNA recovery, and it was used as the normalization control to monitor the effectiveness of RT-PCR and qPCR. First, total RNA containing small RNAs was isolated from 200 μL serum using the miRNeasy Serum/Plasma Kit , and 3.5 μL of the miRNeasy Serum/Plasma Spike-In Control (1.6 ×108 copies/μL working solution) was then added to the plasma samples to extract again (recommended by Qiagen) according to the manufacturer’s instructions. After the addition of a denaturing solution, 3.5 μL of the miRNeasy Serum/Plasma Spike-In Control (1.6 ×108 copies/μL working solution) was added to the plasma samples to extract again (recommended by Qiagen). Eventually, total RNA containing miRNAs was eluted in 14 μL of RNase-free water. A NanoDrop 1000  was applied to quantify the concentration of RNA, reverse transcribed to cDNA and quantitative real-time PCR for specific miRNAs was performed.According to our previous study  and otheAccording to our previous study , the miShttp://www.mirbase.org/. Each 20 μL PCR mix contained 10 μL 2x QuantiTect SYBR Green PCR Master Mix, 2 μL 10x miScript Universal Primer, 2 μL 10x QuantiTect miScript Primer Assay, 2.5 μL RNase-free water, and 3.5 μL cDNA as template. The reactions were incubated at 95°C for 15 min, followed by 55 cycles of 94°C for 15 s and 55°C for 30 s and 70°C for 30 s. All reactions were run in duplicate. The threshold cycle (Ct) was calculated using the second-derivative max method. Ct values greater than 40 were treated as undetermined (- (Ct [miRNA] -Ct [cel-miR-39]).Specific miRNA quantification was performed by SYBR Green-based real-time PCR using a miScript SYBR Green PCR Kit . Detailed information about the six candidates is presented in termined . The relpost hoc multiple comparisons test. The Mann-Whitney U test was performed to compare data that were not normally distributed. The chi-square test was employed to compare gender distributions. Logistic regression analysis was used to detect the risk factors of atherosclerosis in patients with SCH. Spearman’s correlations were performed to explore the associations between TSH, lipid parameters, miRNAs and CIMT, NO, and FMD. GraphPad Prism 8.0.1  was used to perform an agglomerative hierarchical cluster analysis based on the miRNA expression patterns. Receiver operating characteristic (ROC) curve analysis was performed to assess the prognostic ability of candidate miRNAs for arteriosclerosis. All data were processed by the SPSS software package for Windows version 25.0 . All statistical tests were two-sided, and a value of p< 0.05 was defined as statistically significant.Data are presented as the mean ± SEM or raw numbers. The normality of the variables was assessed by the Shapiro-Wilk test. Data from the Kruskal-Wallis test. Normal distribution was analyzed by one-way analysis of variance (ANOVA) and the LSD As displayed in In the qPCR analysis, all six miRNAs and the spike-in control miRNA mimic (cel-miR-39) showed reliable Ct values in all subjects (P = 0.945). The miR-210 critical to hypoxia-induced vascular proliferation showed a slight but insignificant stepwise trend from the NC group (-8.746 ± 1.368) to the SCH group (-8.649 ± 1.041) and then to the SCH+ATH group (-8.239 ± 1.195), the ATH group (-8.156 ± 1.159). Both miR-126-3p and miR-150 showed a significant decreasing trend from the NC group  to the SCH group  and then to the SCH+ATH group , whereas no differences were identified between the SCH+ATH and the ATH groups. Both miR-221-3p and miR-222-3p were significantly downregulated in the SCH+ATH , SCH groups , and ATH groups , but there was no significant difference between the SCH+ATH and ATH groups .As described in As shown in With regard to the six selected miRNAs in our research Table 2,P<0.001), LDL-c  and TC  were positively correlated with ATH. Other parameters including FMD, NO, miR-126, miR-150, miR-221 and miR-222 were negatively correlated with ATH. However, BMI, age, TSH, gender and TG were not found to be significantly correlated with pathological progression of ATH.As shown in Hierarchical clustering analysis can find the linear combinations of miRNAs that maximize the probability of correctly assigning subjects . The expReceiver operating characteristic (ROC) curves were constructed to evaluate the sensitivity and specificity of six candidate serum miRNAs in predicting endothelial dysfunction of all subclinical hypothyroidism patients and atherosclerosis patients. The areas under the ROC curves (AUCs) of the six miRNAs are displayed in In this study, we reanalyzed the association between blood lipid profiles, TSH, CIMT and endothelial dysfunction, including increased NO production and FMD using high-resolution B-mode ultrasound in patients with SCH. Results in our analysis showed that patients in the SCH, SCH+ATH, and ATH groups differed from NC subjects in the mean value of TG, TC, LDL-c, CIMT, FMD and NO. Furthermore, TSH was positively correlated with TC and LDL-c, and was negatively correlated with FMD and NO, suggesting that TSH may be conducive to dyslipidemia, arteriosclerosis and EC injury. This finding is in line with the results of other studies that have proven these associations . With remiRNAs, as key regulators in gene expression networks, can influence many biological processes . IncreasLogistic regression analysis showed that FMD, NO, miR-150, miR-221, miR-222 and miR-126 were negatively correlated with ATH in SCH patients. TC, LDL-c and miR-21 were positively correlated with ATH. Lipid metabolism disorders play a significant role in the pathological progression of ATH, which may be because hyperlipidemia can produce numerous lipid oxides deposited in the vascular endothelium, affected the activation of the coagulation and fibrinolysis systems. resulted in the dysfunction of ECs and accelerating the occurrence of ATH. Patients with elevated serum NO levels and FMD have a significant lower risk of ATH and EC dysfunction. NO is produced in almost all tissues and organs, exerting a variety of biological actions . Many keP< 0.001) and NO , suggesting that miR-126-3p may be involved in the critical targets and pathways of pro-ATH and endothelial dysfunction in patients with SCH. miR-126 is a miRNA that is well characterized to regulate vascular angiogenesis, vascular repair, inflammation activation and apoptosis ; Shandong Provincial Key Laboratory of Endocrinology and Lipid Metabolism (Grant No. SDkeylab-Endo&LiMe2019-01); Natural Science Foundation of Shandong Province of China (Grant No. ZR2017LH023); Medical and Health Project of Shandong Province, China (Grant No. 2015WS0096) as well as Academic Promotion Program of Shandong First Medical University (Grant No. 2019QL017).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Heart transplant (HT) recipients have a high risk of developing severe COVID-19. Immunoglobulin G antibodies are considered to provide protective immunity and T-cell activity is thought to confer protection from severe disease. However, data on T-cell response to mRNA vaccination in a context of HT remains limited.In 96 HT patients, a IFN-γ release assay and an anti-Spike antibody test were used to evaluate the ability of SARS-CoV-2 mRNA vaccines to generate cellular and humoral immune response. Blood samples were collected few weeks to 7 months after vaccination. Multiple fractional polynomial and LASSO regression models were used to define predictors of T-cell response.Three to five months after vaccination, three doses of vaccine induced a positive SARS-CoV-2 T-cell response in 47% of recipients and a positive humoral response in 83% of recipients, 11.1% of patients remained negative for both T and B cell responses. Three doses were necessary to reach high IgG response levels (>590 BAU/mL), which were obtained in a third of patients. Immunity was greatly amplified in the group who had three vaccine doses plus COVID-19 infection.Our study revealed that T and B immunity decreases over time, leading us to suggest the interest of a booster vaccination at 5 months after the third dose. Moreover, a close follow-up of immune response following vaccination is needed to ensure ongoing immune protection. We also found that significant predictors of higher cellular response were infection and active smoking, regardless of immunosuppressive treatment with mycophenolate mofetil (MMF). Solid-organ transplant recipients are at high risk of severe COVID-19, essentially because they are chronically immunosuppressed and frequently suffer from comorbid disease . HT patiIn addition to high rates of complications and mortality from COVID-19 infections, the pandemic has complicated the transplant process. It added new questions regarding donor and recipient screening, potential exposure of recipients to a greater risk of immunosuppression and decision to transplant the patient. Moreover, the clinical implications of COVID-19 infection may differ depending on the type of organ transplanted and the recipient’s comorbidities which further impact decisions to pursue transplantation , 6. Soli+ and CD8+ T-cell responses have also been shown to play important roles in the resolution of SARS-CoV-2 infection  were included. One patient refused any vaccination and was therefore excluded from the analysis. Three patients who refused the third dose were included in the two-dose group. A total of 96 patients underwent a single IgG and IFN-γ release assay testing during hospitalization or clinical follow-up.Blood samples collected at varying times from 1 week to 7 months after vaccination were analyzed. Because patients were included throughout the study period, the number of doses were variable; the occurrence of a COVID-19 infection was pointed out. The institutional review board  approved the protocol, and considered it to constitute routine clinical practice. The need for informed consent was waived, but all patients were given clear information about the study, and their non-opposition was obtained. Collection of nominative data was approved by the national authority for the protection of privacy and personal data.The SARS-CoV-2-specific T-cell response was measured with a commercially available IFN-γ Release Assay  in heparinized whole blood following the manufacturer’s description.+ T cells and Ag 2, covering the S protein that stimulate both CD4+ and CD8+ T cells. Briefly, four blood collection tubes were drawn: Ag1, Ag2, and controls (nil and mitogen). One milliliter of whole blood was incubated for 16–24 h at 37°C, after which plasma was harvested and tested using an enzyme-linked immunosorbent assay (ELISA) for the presence of IFN-γ produced in response to the peptide antigens or mitogen. The nil result was adjusted for background, heterophile antibody effects or non-specific IFN-γ in blood samples. The results were expressed in IFN-γ IU/mL after subtraction of the negative control values as interpolated from a standard calibration curve. The cut-off was 0.15 IU/mL, as determined by the manufacturer.Whole blood was stimulated with SARS-CoV-2 spike antigens during incubation for 16–24 h using two combination of peptides: Ag 1, covering the sub-unit S1 of the spike protein that stimulate CD41 The cut-off value was 50 AU/mL , as determined by the manufacturer. A SARS-CoV-2 IgG concentration of 4,160 AU/mL  considered to be neutralizing at 95% with the ancestral strain , was defined as high in this study.The SARS-CoV-2 IgG II Quant assay is a chemiluminescent microparticle immunoassay that is used for the quantitative determination of IgG antibodies, including neutralizing antibodies, to the receptor binding domain (RBD) of the subunit 1 of the S protein of SARS-CoV-2, using the Architect system (Abbott). Data were expressed in arbitrary units (AU)/mL. Binding antibody units (BAU/mL) were calculated after multiplication by 0.142 on the basis of the results of the World Health Organization International Standard study.Continuous variables were expressed as means ± SEM, and categorical variables as frequencies and percentages. Univariate regression analysis was used to assess the relationship between having Ac > 590 BAU/mL and a positive T-cell response. To study variables associated to T-cell response, two major multivariate regression models were applied to ensure the reliability of the variables we finally selected.The multivariable fractional polynomials (MFP) logistic regressions model was used to investigate non-linear associations between T-cell response and selected variables. Correlations between variables were checked before inclusion in the model in order to avoid collinearity. The MFP model is a method that allows software to determine whether an explanatory variable is important for the model –24. Becap < 0.05. STATA v15  was used for the statistical analyses.The second multi-logistic regression model we used was the least absolute shrinkage and selection operator (LASSO). LASSO regression analysis with EBIC (extended Bayesian information criterion) was performed to achieve enhanced variable selection , 26. ComAmong the 96 recipients included in the study, 95 had a heart transplantation and one had a heart and kidney transplantation. The cohort included a high percentage of male patients (83.3%) with an average age of 58.7 ± 1.28. A vast majority of the patients were older than 40 (91%).n = 26) and diabetes (n = 15); 13 patients had a family history of coronary heart disease . The major reasons for transplant were dilated cardiomyopathy (39.2%) and ischemic heart disease (33%). Half of the patients had a high BMI (>25), and half were active smokers. Almost all patients (97%) were on an immunosuppressive regimen with prednisolone, and about half (45%) were on mycophenolate mofetil (MMF) combined with an mTOR inhibitor (everolimus or sirolimus). Previous medical history included hypertension ( disease .2 Although about three quarters (74%) of patients had lymphopenia, 27 (28.1%) patients had a positive result for Ag1 and 39 (40.6%) had a positive Ag2 (IFN-γ ≥ 0.15 IU/mL) , four patients received heterologous vaccination with mRNA and recombinant vaccine, one in the two doses group, four in the three doses group . Data on5 IU/mL) . A positp < 0.01 by Kruskal-Wallis test), however, analysis with logistic regression showed that neutrophils count was not a significant predictor of a positive T-cell response in univariate analysis.Patients had more neutrophils in the smoker group compared to the non-smoker group , 22 of them had IgG > 590 BAU/mL.Patients positive for Ag1 were all positive for IgG. Among the 96 enrolled patients, about one third were positive for Ag1, Ag2, and IgG, T-cell responses were detected in 20% (5/25) of seronegative subjects.Seropositive responses were detected in 63.6% (35/55) of negative T cell subjects, but among these, 7.2% (4/55) had antibodies at a high level (>590 BAU/mL).p = 0.000).Most of the patients with IgG > 590 BAU/mL were T responders [81.8% (59.7–94.8%)]. Analysis with logistic regression showed that Ac > 590 BAU/mL was an significant predictor of a positive T-cell response in univariate analysis , the IgG response , the time elapsed since the last vaccine dose , the occurrence of a SARS-CoV2 infection , and the lymphocyte count . In addition, active smoking tended to be significant . We did a bootstrap for internal validation, the results of the MFP and the bootstrap were reliable, except for IgG response, which was no longer significantly associated. Active smoking was significantly associated with IFN-γ release after bootstrapping . The adjusted R-squared value of MFP model was 0.683, meaning that the significant variables explained 68.3% of the T-cell response . Multivariable analysis using MFP revealed that each Ag1 and Ag2 response was independently associated with: age  was associated with T-cell activity (Ag2) in vaccinated patients with HT as well as the time lapse between dosage and vaccination (p < 0.023), the occurrence of an infection (p < 0.000) and smoking (p < 0.03). However, sex was not independently associated with Ag2 (p = 0.07). The adjusted R-squared value of the regression based on LASSO2, was 0.52, which means that 52% is the fraction of the T-cell response that is predicted by these independent variables  in vaccinated HT patients are occurrence of an infection and active smoking.n = 71). Mean values for T-cell response (Ag1 and Ag2) were above threshold from 3 to 5 months following the vaccine (mean of 0.20 0.06 for Ag1 and 0.31 ± 0.16 for Ag2) (We first focused on patients that had received three doses of vaccine (for Ag2) . Half offor Ag2) . In the None of the patients from the two-dose group had high levels of antibodies (>590 BAU/mL). In the three-dose group, high antibodies levels were detected in 36.1% of the patients from 3 to 5 months after the last dose (13/36). In the small group 4including seven patients who had had three doses plus COVID-19 infection and one patient that received with four doses, all patients had high antibodies levels and positive T-cell response regardless of the time since the last vaccine dose (1–5 months).Thus, a third vaccine dose generated a great anti-vaccine response in HT patients from 3 to 5 months post vaccination, and it also provided a proportion of nearly 50% of humoral and cellular responders.Immune response was far stronger in the group 4, probably because supplemental stimulation occurred via the natural infection for 7 out of 8 patients. From 3 to 5 months post vaccine, the mean IgG level reached 4456.1 BAU/mL, and mean Ag2 was 1.82 IU/mL .Our investigation of immune response to mRNA vaccine in a large cohort of HT patients in the post vaccination period showed that humoral response was elicited more frequently than T-cell response.COVID-19 severity varies widely, ranging from asymptomatic infection to severe pneumonia and multi-organ system dysfunction that can lead to the death. Overall, we reported a T cell response in 40.6% of HT patients, and nearly half of the HT patients who had received a third dose of the vaccine had a positive cellular response at a time-lapse of 3–5 months after the last vaccination. In previous studies, T cell response in HT subjects was measured only shortly after vaccination (2–3 weeks) . It was In previous studies, after two vaccine doses, the humoral immune response to the BNT162b2 vaccine was found to wane over a period of 6 months in HT patients , 29. We A surprising finding of our study was that active smoking was statistically associated with quantitative T-cell response, which has not been previously reported. Many studies reported that smokers appeared to be protected against SARS-CoV-2 infections, while other studies underlined an increased risk for severe COVID-19 in heavy smokers. The effect of cigarette smoking on COVID-19 is still controversial  and the Not as reported for the humoral response in HT recipients , and morOur study also underlines the good immune response induced by infection in the small group of patients with a COVID-19 infection plus three doses, since all individuals had high IgG and T-cell response. Previous COVID-19 infection enhanced also magnitude and longevity of the humoral response following vaccination with mRNA vaccines of adult patients on hemodialysis or SOTs , 47. SimHaving 3 vs. 2 or zero doses of mRNA vaccine was associated with increased protection of the general population against both the Omicron and Delta variants . BoosterSome side effects of SARS-CoV-2 vaccines, such as fever, headache, fatigue and pain at the injection site have been described. Organ rejection post-COVID-19 vaccination is rare but can occur with all SARS-CoV-2 vaccines or following COVID-19 infection. However, only one case of heart rejection among 136 cases was described by Alhumaid et al. . The smaWe performed T cell analysis using QuantiFERON assay, that does not need to isolate PBMC. The same volume of blood is drawn, the T cell response is evaluated by mL of blood and can be compared between patients. Alternatively, cytokine-producing cells may be enumerated using an ELISPOT assay or flow cytometry-based intracellular cytokine staining. ELISPOT gives the IFN-γ cell concentration per T cell. In the context of tuberculosis disease (TB), the QFT-TB assay lacks of sensitivity with many indederminate results. These are related to an alteration of the mitogen response  and are One limitation in our study is the heterogeneity of the group. Assays were done at the time of clinical follow up, and IgG and T-cell activity was assessed once rather than repeatedly, so we were not able to perform a longitudinal analysis. On the other hand, the post-vaccination study period was long enough to observe the waning of humoral and cellular immunity.Overall, studies on T-cell response in HT are currently sparse. Our study provides novel data on the immunogenicity of COVID-19 mRNA vaccines on a large cohort of HT patients, in particular on T cell response. Our findings underline the interest of booster vaccinations among HT recipients, who are a population at risk of severe disease, and we suggest the importance of follow-up for antibody and T-cell response following vaccination in order to ensure sufficient immune protection.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The need for informed consent was waived, but all patients were given clear information about the study, and their non-opposition was obtained.J-BB, CA, and SG involved with study design. AC, SG, and CM contributed to patient data acquisition. L-SA-G and CM made the statistical analysis. CM wrote the manuscript. AC, J-BB, CA, L-SA-G, and SG revised the manuscript. All authors contributed to the article and approved the submitted version.


Given its cytostatic effect, studies conducted in the 1990's showed that paclitaxel was toxic to many pathogenic fungi and oomycetes. Further studies led to the idea that the differences in paclitaxel sensitivity exhibited by different fungi were due to differences in the β-tubulin protein sequence. With the recent isolation of endophytic fungi from the leaves and bark of the Himalayan Yew, Taxus wallichiana Zucc., and the availability of genomes from paclitaxel-producing fungi, we decided to further explore the idea that endophytic fungi isolated from Yews should be well-adapted to their environment by encoding β-tubulin proteins that are insensitive to paclitaxel. Our results found evidence of episodic positive/diversifying selection at 10 sites  in the β-tubulin sequences, corresponding to codon positions 33, 55, 172, 218, 279, 335, 359, 362, 379, and 406. Four of these positions  have been implicated in the binding of paclitaxel by β-tubulin or formed part of the binding pocket. As expected, all the fungal endophytes grew in different media regardless of the paclitaxel concentration tested. Furthermore, our results also showed that Taxomyces andreanae CBS 279.92, the first fungus shown to produce paclitaxel, is a Basidiomycete fungus as the two beta tubulins encoded by the fungus clustered together with other Basidiomycete fungi.Paclitaxel, better known as the anticancer drug Taxol The radial growth rate (Kr) was calculated as defined by Trinci (1-radius0) / (time1-time0)  were used to determine the effects of the paclitaxel treatment on the mycelia growth rate.A one-way analysis of variance (ANOVA) test and Duncan Agar plugs from fungi , grown igDNA was extracted using the NucleoSpin Plant II kit  by following their protocol. The internal transcribed spacer (ITS) using primers ITS1F (5'-CTT GGT CAT TTA GAG GAA GTA A-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') was amplified and sequenced to identify the endophytic fungi at the molecular level . Plasmids were isolated using the GeneJET Plasmid Miniprep Kit (Thermo Scientific™) and sent to Macrogen Europe (The Netherlands) for sequencing using the universal primers provided by the facility.Polymerase chain reactions were done using Phusion Hot Start II DNA Polymerase (Thermo Scientific™) following the manufacturer's protocol, but annealing temperatures were carried at 59°C and 58°C for the beta-tubulin gene and ITS sequence, respectively. PCR products were gel-purified using the GeneJET Gel Extraction Kit (Thermo Scientific™), cloned into pJet1.2 (Thermo Scientific™), and transformed into One ShotTaxus spp. or from fungi deemed able to synthesize paclitaxel were included in the analysis  was used to test for episodic positive/diversifying selection, but the analysis was limited to those organisms isolated as endophytes from Taxus spp. or able to produce paclitaxel.The β-tubulin genes sequences were translated, and the Molecular Evolutionary Genetics Analysis (MEGA) software was used to align and analyze the protein sequences. The analysis only included the 428 amino acids that have been implicated in the paclitaxel-β-tubulin interaction. In addition, GenBank protein sequences from fungal endophytes isolated from analysis . For comH. annosum, grew in both media containing different concentrations of paclitaxel  (H. annosum was higher compared to when it was grown in PDA (P < 0.001)  and B. adusta strain PAR7 (dikaryon), showed a significantly higher GA than H. annosum, regardless of the media used (P < 0.001)  . The Annulohypoxylon sp. strain MUS1, known to produce paclitaxel, showed an identical GA as A. arborescens strain PAR15 irrespective of the medium, and it showed a slight but significant (P < 0.05) increase in the GA when it was grown in MMN (r) and when grown in either medium, the Kr remained relatively constant regardless of the concentration of paclitaxel used (r of H. annosum was significantly lower when grown in PDA (P < 0.001), while Diaporthe sp. strain PAR5 showed a decrease in the Kr when grown in MMN. Though not significant, B. adusta strain PAR4 (monokaryon) and B. adusta strain PAR7 (dikaryon) showed a slight increase in the Kr when grown in MMN. Similarly, Annulohypoxylon sp. strain MUS1 showed a slight increase in the Kr when grown in MMN. All the endophytic yeasts isolated  were able to grow at all the different concentrations of paclitaxel tested (data not shown).All the endophytic fungi, as well as ctively) . When gr< 0.001) . Though nificant . The Bas< 0.001) . Alterna< 0.001) . Though n in MMN . The funxel used . HoweverM. guilliermondii strain MUS2. The accession numbers are included in A partial β-tubulin coding-sequence was successfully amplified for the 14 fungal endophytes chosen. The β-tubulin sequence and the ITS sequence for each fungus were deposited in GenBank. However, a different forward primer (ATG AGA GAA ATT ATT CAC TTG) was used to amplify the β-tubulin for Taxus spp. and the most highly expressed human β-tubulin (P07437) clustered together in a separate branch with the rest of the Basidiomycete β-tubulins. Surprisingly, the Taxomyces andreanae β1-tubulin also clustered in this group, and the β2 tubulin clustered with the rest of the β2 tubulin from Basidiomycete fungi in a separate clade. This would suggest that T. andreanae CBS 279.92 belongs in the phylum Basidiomycota, rather than Ascomycota as originally proposed. The β-tubulin from the endophytic yeasts  clustered together with S. cerevisiae and was similar at four out of the five key amino acids  initially mutated to create a paclitaxel-sensitive yeast (2- and β1-tubulins distributed among the clades. The β2- or β1-tubulins from paclitaxel-producing fungi (indicated with an asterisk) did not cluster together  in the β-tubulin genes, corresponding to codon positions 33, 55, 172, 218, 279, 335, 359, 362, 379, and 406. Five of these positions  have been implicated in the binding of paclitaxel by β-tubulin  was used to analyze sequences from fungal endophytes isolated from -tubulin .P. microspora Ne32, identified two regions in the β-tubulin protein involved in the binding of paclitaxel, with amino acid residue 219 being a Thr for susceptible fungi or Asn or Gln for resistant fungi . Based on their calculations, mutations at positions 24, 25, 26, 229, 280, and 359 would likely affect the binding energy of paclitaxel to β-tubulin, and later, this was validated in part by Kudo et al.  yielded the second β-tubulin gene, a partial 5.S RNA sequence, and both the small (SSU) and large subunit (LSU) ribosomal RNA genes. Based on comparisons of the SSU and LSU sequences, T. andreanae CBS 279.92 should be placed under the Phanerochaetaceae family and most likely belongs in the genus Ceriporiopsis, with C. gilvescens being the closest relative (data not shown). We were unable to obtain T. andreanae CBS 279.92 from the culture collection (Westerdijk Institute) to further validate our findings by ITS sequencing since the fungus is not available from their public collection . In addition, we were surprised to see that isolate PAR4, chosen before the ITS and β-tubulin genes were amplified and sequenced, was another isolate of Bjerkandera adusta. Unlike B. adusta strain PAR7, strain PAR4 did not form clamp connections in culture. When looking at the sites 24, 25, 26, 229, 280, and 359, which were mentioned by Tuszynski et al.  formed distinct clades along the Ascomycete and Basidiomycete divisions . Interesi et al. , no cleawww.datamonkey.org) was used to analyze sequences from fungal endophytes isolated from Taxus spp., as well as organisms shown to synthesize paclitaxel. MEME, which uses a mixed-effects maximum likelihood approach looking for individual sites under positive selection  in the β-tubulin sequences, corresponding to codon positions 33, 55, 172, 218, 279, 335, 359, 362, 379, and 406. Four of these positions  have been implicated in the binding of paclitaxel by β-tubulin or formed part of the binding pocket [ were able to grow at all the paclitaxel concentrations . Though we do not dispute the findings of Soliman et al., we contend that the wood-decaying fungi used in their study  are mainly root and butt rot-causing pathogens. In fact, our department has worked with Heterobasidion species for more than 20 years, and PDA is never used for culturing this fungus. Though paclitaxel growth assays conducted since the 1990s have only used PDA media   can be found in the article/HV conceived, designed, performed the experiments, and wrote the manuscript. DG provided the fungal isolates. MG-G contributed to the reagents and materials. HV and MG-G analyzed the data. All authors read, edited, and approved the manuscript.This work was financially supported by a grant from the Swedish Research Council (Ref/348-2012-6138/Agreement/C0613801) to MG-G and DG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pathogenic organisms and antibiotic-resistant strains were detected in the DR groups (DM-NPDR and DM-PDR). The NGS analysis showed that potentially pathogenic bacteria such as Enterobacteriaceae, Neisseriaceae, Escherichia-Shigella, and Pseudomonas predominated in DM, especially in DR. There was dissimilarity in the ocular surface microbiome between DM and non-DM groups. The subgroup analysis showed that the DR group had significantly different microbial community from DM-no DR and non-DM groups . The microbial community in the poorly controlled DM was also significantly different from well-controlled DM and non-DM groups (p < 0.001). Using the NGS method, our study is the first to signify the importance of DR and glycemic control status, which affect the changes in the ocular surface microbiome.This cross-sectional, age- and gender-matched study included 20 eyes of non-diabetic subjects (non-DM group) and 60 eyes of type 2 diabetes mellitus (DM group). Subgroups of DM were classified by diabetic retinopathy (DR) staging into no DR (DM-no DR), non-proliferative DR (DM-NPDR), proliferative DR (DM-PDR), and by glycemic control . Conjunctival swabs were performed for ocular surface microbiome analysis using conventional culture and next-generation sequencing analysis (NGS). A higher culture-positive rate was found in DM (15%) than in non-DM group (5%) ( In Thailand, the estimated number of DM may reach 4.3 million in 20352. The most common eye complication in DM patients is diabetic retinopathy (DR), which is the leading cause of blindness worldwide2. Other ophthalmic complications include cataract, glaucoma, and ocular surface changes4.Diabetes mellitus (DM), a metabolic disorder characterized by disturbance of glucose metabolism and chronic hyperglycemia, is a significant threat to global health. The prevalence of DM has increased rapidly over the past few decades and is estimated to rise from 463 million in 2019 to 700 million in 2045 worldwide6. Previous studies revealed that such changes were more commonly associated with type 2 DM and proportional to the severity of DR and HbA1c level9. Moreover, DM increases the risk of infection such as conjunctivitis, corneal ulcer, and endophthalmitis14 by altering ocular protective immune response, including decreased cytokine production and impaired cellular immune response functions17. Alterations of immune function, together with ocular surface changes in DM, may disturb the normal ocular surface microenvironment and microbiome. Moreover, the common treatment modalities in the advanced stage of DR are intravitreal drug injection or intraocular surgery. These procedures may introduce the ocular surface microbes into the eye, resulting in intraocular infection18. The previous reports showed more bacterial colonization and the presence of gram-negative bacteria on the ocular surface of the DM than in the non-DM groups23. Consistently, the most common organisms causing postoperative endophthalmitis reported in DM patients were coagulase-negative Staphylococcus and gram-negative bacteria, which correlates with conjunctival flora of the DM24.The ocular surface changes in DM patients include tear film dysfunction, increased conjunctival metaplasia, decreased conjunctival goblet cell density, decreased sub-basal nerve density, decreased corneal sensitivity, and delayed epithelial and stromal wound healing25. Therefore, the development of advanced technology using next-generation sequencing (NGS) has tremendously contributed to the current research. This high-throughput method can generate a million reads per run, thus reducing the cost and analytic time28. Many studies have reported the changes in the ocular surface microbiome in DM using the culture technique23, but only a few applied the NGS method32.The culture method is the gold standard for detecting microorganisms. This conventional technique has significant limitations when studying the ocular surface microbiome due to the detection of fastidious microorganisms and small amounts of specimensTo our knowledge, there is no previous study on the ocular surface microbiome in different stages of DR and the level of glycemic control using the NGS method. Therefore, we conducted a study to identify the difference in the microbial community between DM and non-DM groups using the NGS method and determine the possible effects of the DR severity and glycemic control on the ocular surface microbiome. Results from this study may help improve the understanding of the pathogenesis underlying the increased risks of ocular infections in DM.A total of 80 eyes (80 subjects) included 60 eyes of DM and 20 eyes of non-DM subjects. The DM group was further classified into 3 subgroups according to diabetic retinopathy (DR) staging as no diabetic retinopathy (DM-no DR), non-proliferative diabetic retinopathy (DM-NPDR), and proliferative diabetic retinopathy (DM-PDR) (20 eyes/subgroup). The demographics and clinical details are shown in Supplementary Table 1. The mean age of all subjects was 55.6 years old (range 37–79 years old), and 50% were male. Most of the subjects were office workers who resided in the central area of Thailand. The DM subjects were also classified by glycemic control into 2 subgroups, well-controlled DM (HbA1c < 7%) and poorly controlled DM (HbA1c ≥ 7%), which was the majority (60%). The duration of DM ranged from 4 months to 36 years. Among subjects with DR, 13 eyes had diabetic macular edema, and 12 eyes had vitreous hemorrhage.p value = 0.437). There were no significant differences in the culture-positive rate among non-DM and the three DM subgroups . Seven microorganisms were identified, mostly gram-positive cocci, which Staphylococcus epidermidis being the most common. In addition, Providencia rettgeri was isolated from the DM-NPDR subgroup, while Kocuria palustris and Micrococcus luteus were isolated from the DM-PDR subgroup. There was no significant difference in the culture-positive rate between well-controlled and poorly controlled DM subgroups .A higher culture-positive rate was shown in the DM (15%), compared to the non-DM group (5%), without significant difference  was isolated from the DM-PDR subgroup. Antibiotic-resistant microorganisms were found in both well-controlled and poorly controlled DM subgroups. The details of the culture and antibiotic susceptibility are shown in Table Antibiotic-resistant microorganisms were only found in the DM-NPDR and DM-PDR subgroups. Resistance to benzylpenicillin, erythromycin, clindamycin, and fusidic acid in Illumina sequencing of 16S rRNA genes generated 3,698,312 total reads. After quality data processing, 1,786,948 high-quality reads were obtained. There was an average of 22,337 reads per sample . The number of amplicon sequence variants (ASVs) was 1359. The approximate saturation of microbial richness of all samples was 4534 sequencing depths, as estimated by the rarefaction curves. The plateau curve in rarefaction was observed when approximately 2000 sequencing depths were reached.The 100% core ASVs sample matching was used to identify the core ocular surface microbiome. At the phylum level, Proteobacteria, Firmicutes, and Actinobacteria were the core microbiome of the non-DM group, and Proteobacteria and Firmicutes of the DM group. At the class level, Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, and Bacilli were the core microbiome of the non-DM group, and Gammaproteobacteria and Bacilli of the DM group.p value = 0.021) , while Gammaproteobacteria was significantly abundant in the DM group . At the family level, Enterobacteriaceae was significantly more abundant in the DM than non-DM groups . Neisseriaceae was significantly abundant in the DM-NPDR compared to the DM-no DR and non-DM groups . At the genus level, Escherichia-Shigella was significantly abundant in the DM compared to the non-DM group   compared to the DM-NPDR (2.35%), DM-no DR (2.42%), and non-DM group (3.05%), with no statistical significance   and poorly controlled DM (10.02%), compared to the non-DM group (5.39%)  , followed by Firmicutes (32.43% and 34.83%), Actinobacteria (13.43% and 10.71%), and Bacteroidetes (9.35% and 6.71%)  was performed to determine the significant difference in the bacterial distribution between groups. The bar plot represents the effect size LDA  for a significant taxon in a particular group. Bacterial taxa with LDA scores greater than 2 were considered significant. The results are shown in Fig. p value > 0.05). For the subgroups of DM classified by DR staging and glycemic control, there was no statistically significant difference in the alpha-diversity indices between each subgroup. The box plots of the Shannon diversity index are demonstrated in Supplementary Figure 1.Alpha-diversity was performed using observed ASVs, Chao1, Shannon, and phylogenetic diversity (PD) whole tree. All analyses showed no significant difference between the non-DM and DM groups  . However, there was no significant difference in the microbial community within the DM-NPDR and DM-PDR subgroups, as well as in the DM-no DR and non-DM groups    using unweighted UniFrac distance showed that the human ocular surface microbial community in the DM group was significantly different from the non-DM group . The lower culture-positive rate in our study may be due to different specimen collection and culture techniques23.This study is the first to report the ocular surface microbiome in DM according to DR staging and glycemic control using the NGS method. Our results signify the effect of these factors on the ocular surface microbial community. Previous studies described the ocular surface microbiome in DM, mainly using conventional culture technique. The culture-positive rate was shown to be higher in DM than non-DM group Staphylococcus. Gram-negative bacteria were more commonly found in the DM group, consistent with prior reports20. Furthermore, the colonization of pathogenic bacteria was more detected in the DM than in the non-DM group21. P. rettgeri, which can be found in normal healthy gut but can lead to opportunistic infection, was found in the DM-NPDR group in this study. Koreishi et al.33 reported that P. rettgeri was a potential microorganism causing ocular infections such as conjunctivitis, keratitis, dacryocystitis, and endophthalmitis in immunocompromised patients, including DM. Kocuria spp., gram-positive bacteria found in the DM-PDR group, was reported to cause canaliculitis, dacryocystitis, recurrent conjunctivitis, and severe marginal keratitis with corneal melt35.Most microorganisms identified were gram-positive cocci with mainly coagulase-negative 20 reported that DM with DR had more culture-positive rate than DM-no DR subjects. However, this study found no significant difference in culture-positive rate when subgroup analysis by either the DR severity or glycemic control was done. Remarkably, this study detected the antibiotic-resistant organisms only in DM with DR group (including P. rettgeri and S. hominis in the DM-NPDR group and MRSE in the DM-PDR group). One possible explanation for this finding is that DM with DR subjects is likely to require intraocular intervention and, as a consequence, exposed to antibiotics36. For instance, several studies reported the emergence of antibiotic-resistant organisms after multiple sessions of intravitreal drug injection using prophylactic antibiotics38.Martins et al.32. At the class level, we found that Alphaproteobacteria predominated in the non-DM group, and Gammaproteobacteria predominated in the DM group, similar to the reported from Ham et al.29 Prior studies have demonstrated the abundance of potentially pathogenic bacteria in the DM group, using NGS technique32. Our study likewise found a higher proportion of Enterobacteriaceae, Neisseriaceae, Escherichia-Shigella, and Pseudomonas in the DM group, especially in DM with the DR group.The NGS analysis showed that the top four most common phyla in both non-DM and DM groups were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes, which were consistent with previous studies32. Nevertheless, our study using 4 indices did not confirm such findings. The dissimilarity may be from the different sample collection techniques, analysis, and populations. However, from the beta-diversity analysis, we can first demonstrate that the presence of DR and poorly controlled DM status can significantly change the microbial community on the ocular surface. Many groups have reported more severity of ocular surface changes in DR and poorly controlled DM9. As the level of advanced glycation end product (AGE) is significantly increased in patients with DM-PDR39. This level affects the ocular surface by reducing proliferation, enhancing apoptosis of epithelial cells, and inducing inflammation in the sub-basal nerve plexus6. Together with the alteration of protective immune response in DM17, these can lead to ocular surface microbiome dysbiosis and increase the abundance of potential pathogens in DM, especially in the DR subgroup.The ocular surface microbiome was previously reported to be more diverse in alpha-diversity in the DM compared to the non-DM groupThe limitations of this study were small sample size and 16S rRNA incapability to detect viruses and fungi, which may be a part of the ocular surface microbiome. In addition, the data collection in a single time point may not represent the whole picture of ocular surface microbiome change with time. Further studies are undoubtedly warranted to understand the cause-and-effect relationship. Additional assessments in the metagenomics profiling, protein expression, and metabolic activity may lead us to a better understanding of ocular surface microbiome change in this significant and common disease.This study demonstrated definite differences in the ocular surface microbiome between the DM and non-DM groups, the DM with DR and no DR groups, and finally, poorly controlled and well-controlled DM groups. In addition, a higher prevalence of pathogenic bacteria and antibiotic-resistant strains was more commonly found in DR compared to the other groups. These results may provide further knowledge on ocular surface diseases and the pathogenesis of many infectious diseases related to the ocular surface microbiome in DM patients.The study was conducted in accordance with the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board, Faculty of Medicine, Chulalongkorn University (IRB No. 377/64 and COA No.704/2021). In addition, the study was registered to the Thai Clinical Trial Registration (TCTR No. TCTR20210427010).This cross-sectional study was conducted at King Chulalongkorn Memorial Hospital (KCMH), Bangkok, Thailand. The inclusion criteria were subjects aged over 18 years old with or without diabetes mellitus. Exclusion criteria were any subjects with the following conditions: having ocular surface diseases; history of ocular and periocular infection/inflammation; having allergic conjunctivitis; previous contact lens used; history of ocular medication, systemic antibiotic, steroid, or immunosuppressive drug use; history of intravitreal drug injection or ocular surgery within the past three months. This study also excluded the subject, who was a healthcare worker.30. From February to March 2022, 20 eyes of 20 non-diabetic subjects (non-DM group) with normal blood sugar or HbA1c tests documented within 1 year and 60 eyes of 60 subjects with a known diagnosis of type 2 diabetes mellitus by ADA criteria40 (DM group)  were recruited. The DM group was further classified by DR staging41 into three subgroups: DM-no DR, DM-NPDR, and DM-PDR groups (20 eyes per group). All groups were age- and gender-matched. This same group was also divided into well-controlled DM (HbA1c < 7%) and poorly controlled DM (HbA1c ≥ 7%). Written informed consent was obtained from all subjects before enrollment.A sample size of 20 eyes per group was calculated based on a formula comparing two independent means and standard deviation from the previous studyThe subjects were interviewed for the demographic data, the Ocular Surface Disease Index (OSDI) questionnaires, and received complete ophthalmic examinations. The conjunctival swab was done before any examinations that touched the ocular surface and the instillation of any drugs, except for topical anesthesia. The processes of conjunctival swab, culture, DNA extraction, and 16S rRNA sequencing processes were done by the masked investigators.The conjunctival swab was collected from one eligible eye of each subject. If both eyes were eligible, only the right eye was included. One drop of 0.5% Tetracaine Hydrochloride solution  was applied to the inferior conjunctival fornix of the selected eye. Three minutes later, the subject’s lower eyelid was pulled down to avoid touching the lid margin or eyelashes, and a sterile cotton swab was swept gently on the inferior conjunctival surface from nasal to temporal 5 times without touching eyelids or eyelashes (rotating the sterile cotton swab each time for 360° of swab collection). Then, the swab was placed in 1 ml phosphate buffer saline (PBS) as a sterile transport medium and sent to the microbiology laboratory. All conjunctival swabs were done by the same investigator. A sterile cotton swab was placed in transport media without swabbing (blank swab) and used as a negative control.2 incubator at 37 °C, 5% CO2, and checked daily for bacterial growth for a week. Bacterial identification was determined by an automated identification system (VITEK 2 XL) and mass spectrometry microbial identification .The sterile pipette was used to transfer the sample onto a chocolate agar plate (approximately 0.1 ml). The plate was incubated in a COThe antibiotic susceptibility test was determined using the Kirby-Bauer disk diffusion method and the VITEK 2 XL system. The antibiotics included benzylpenicillin, ampicillin, oxacillin, piperacillin/tazobactam, cefazolin, cefuroxime, cefuroxime axetil, ceftriaxone, cefepime, ertapenem, imipenem, meropenem, amikacin, gentamycin, ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, erythromycin, clindamycin, chloramphenicol, linezolid, teicoplanin, vancomycin, tetracycline, tigecycline, fusidic acid, rifampicin, and trimethoprim/sulfamethoxazole. The results were interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines.Bacterial DNA extraction was performed using the QIAamp DNA Microbiome Kit , following the manufacturing protocol. DNA was subjected to 16S metagenomic sequencing library preparation. All samples were processed in a single sequencing run.16S rRNA gene was amplified using 341F and 805R primers, targeting V3-V4 variable regions and 2X sparQ HiFi PCR Master Mix . The PCR amplification included an initial denaturation step of 2 min at 98 °C, followed by 30 cycles of 98 °C for 20 s, 55 °C for 30 s, and 72 °C for 1 min, followed by a final extension step at 72 °C for 1 min for one time. Subsequently, 16S amplicons were purified using sparQ Puremag Beads  and indexed using 2.5 µL of each Nextera XT index primer in a 25 µl PCR reaction, followed by 10 cycles of PCR amplification condition above. The final PCR products were cleaned, pooled, and diluted to the final loading concentration at 4 pM. Cluster generation and 250-bp paired-end read sequencing were performed on an Illumina MiSeq  at the Omics Sciences and Bioinformatics Center .https://benjjneb.github.io/dada2/). The microbial diversity and community structures using unique amplicon sequence variants (ASVs) were described by the DADA2 pipeline42. Microbial taxa were classified from Silva version 138 as a reference database43. Alpha-diversity index  was computed using DADA2 software. For Beta-diversity, principal coordinate analysis (PCoA) of unweighted UniFrac distances were plotted from Phyloseq data. Linear discriminant analysis effect size (LEfSe) was performed to identify the bacterial biomarkers.The raw sequences were categorized into groups based on the 5’ barcode sequences. The sequences were processed following the DADA2 v1.16.0 pipeline  or Kruskal–Wallis test was used for comparing more than two groups. Pairwise comparison of alpha-diversity  was calculated using the Kruskal–Wallis test. Permutational multivariate analysis of variance (PERMANOVA) was performed to evaluate the differences in beta-diversity among groups. Moreover, the Kruskal–Wallis sum-rank test was also used in LEfSe analysis to identify bacterial biomarkers that differed significantly in abundant taxon between sample groups. p value less than 0.05 was considered statistically significant.Demographic data were analyzed using descriptive statistics. The Chi-square test was used for comparing categorical data variables between groups. Independent Supplementary Information.


Cold plasma has been considered a novel non-thermal processing technique and attracted a high attention by the food industry. In this study, the influences of dielectric barrier discharge cold plasma (DBD-CP) on the myoglobin (Mb)-added washed pork muscle (WPM) were evaluated. The electrophoresis pattern, autoxidation, and secondary structure of Mb were analyzed. The results found that DBD-CP caused the decrease of the redness and total sulfhydryl (T-SH) in WPM, while the increase of non-heme, peroxide value (PV), and thiobarbituric acid reactive substances (TBARS), suggested that treatment triggered protein oxidation and heme degradation. Additionally, DBD-CP treatment enhanced the autoxidation of Mb, induced the release of intact heme from the globin, rearranged the charged groups, and promoted Mb aggregation. The transformation of α-helix into the random coil of Mb demonstrated that DBD-CP weakened the tensile strength. Overall, data indicated that DBD-CP promoted autoxidation and changed the secondary structure of Mb, accelerating Mb-mediated lipid oxidation in WPM. Thus, further studies about the optimization of processing conditions by DBD-CP need to be performed. During meat processing and storage, lipid oxidation is the main reason causing the deterioration of meat or relevant products, which easily leads to adverse effects on the color attributes, flavor, taste, nutrients, and safety . TherefoDielectric barrier discharge cold plasma (DBD-CP) is a novel processing technology used in agricultural products, which has drawn widespread attention and concern in the food industry because of its energy-saving and environmental characteristics . Cold plLongissimus Dorsi muscle after treatment for 10 min (2+) Mb or ligands such as O2 and NO bind the iron atom of the heme ring moiety of reduced Mb affects the onset of Mb oxidation (Myoglobin (Mb) and hemoglobin (Hb) are the main heme proteins in muscle food, which contribute to the redness of meat. They are also essential endogenous factors that induce lipid oxidation in the muscle. For red muscles, Mb is the main heme protein accounting for more than 50–70% of total heme protein in pork muscle and 65–90% of total heme protein in beef muscle , and lowr 10 min . A coordxidation . BecauseHerein, this study investigated the addition of Mb to washed pork muscle under different DBD-CP treatments . The various indexes of meat samples, including surface color, non-heme iron content, total sulfhydryl, and lipid oxidation were determined during storage. Meanwhile, changes in the structure of Mb treated with DBD-CP were explored by analyzing the Mb oxidation, polyacrylamide gel electrophoresis, absorption spectra, particle size, zeta potential, and circular dichroism spectra.The myoglobin  used in this study was extracted from equine skeletal muscle. Since Mb was unstable and prone to be oxidized, highly oxidized Mb (>95% of metmyoglobin) would affect the experimental process. Therefore, Mb was reduced by sodium hydrosulfite solution under the atmosphere filled with nitrogen . The redFresh pork longissimus lumborum (LL) was purchased from a local market, immediately shipped to the laboratory under cold storage. Washed pork muscle (WPM) was prepared as described formerly . Before According to the previous method, myoglobin was added to washed pork muscle, then stirred well with a glass rod with minor changes . BrieflyThe DBD device used in this paper was the same as Huang et al.  describe*) values of washed pork muscle were determined by Chroma meter . This instrument was adjusted with a white correcting board before measuring. Six different points were selected from the sample for determination, and the results were averaged.CIE-Lab redness  was added to a chloroform/methanol mixture , then homogenized for 40 s using a Polytron Type PT 10/35 (Brinkmann Instruments) at 9000 rpm and poured into a tube . This Po532nm-A600nm was taken as the actual absorbance of the samples. The standard curve of 1,1,3,3-tetramethoxypropane was used to calculate TBARS.TBARS is one of the crucial indicators to judge the secondary oxidation of lipid. According to the method described by Richards et al. , sample ·g−1 of muscle.Total sulfhydryl content was evaluated by Total Sulfhydryl Group Content Assay Kits  . Specifically, WPM (0.1 g) was added into the extracting solution (1 mL) and centrifuged at 8,000 g for 10 min. The supernatant (40 μL) and reagent one (150 μL) were added to the test and control tube, respectively. Then reagent two (10 μL) was added to the test well, 10 μL deionized water was added to the control well and incubated at 25°C for 10 min. A standard curve of glutathione was established according to the absorbance of 412 nm to calculate the concentration. The total sulfhydryl content was expressed as μmolA 10 mL Mb solution prepared as described in Section 2.1 was placed in a petri dish with a diameter of 50 mm and then subjected to DBD-CP treatment. DBD-CP treatment was performed at 0, 50, 60, and 70 kV in air atmospheric for 180 s at room temperature. The operation frequency of the system was 50 Hz. After processing, the Mb solution were rapidly placed at 4 °C for further experiments.According to Luo et al. , the SDSSoret band analysis of Mb solution treated with or without DBD-CP was performed. Briefly, the absorption spectra at 350–700 nm were determined by UV-2600 spectrophotometer . All tests were repeated three times at room temperature.kox) is the rate of metMb generated. The UV-2600 spectrophotometer  was used to record the absorption spectra of Mb in the range of 500–700 nm. The determination method is slightly modified according to the previous study (R1 – 0.085 R2 + 1.262 R3 – 0.520.Mb automatic oxidation rate (us study . Specifius study  as follokox) of Mb is expressed by the slope of metMb% production to time.The automatic oxidation rate . All tests were fulfilled in triplicate at 25°C.CD spectroscopy was evaluated using the method described by Xia et al. . Mb solup < 0.05).The same batch of pork LL was used to prepare all the WPM in the study, and all determinations were carried out in triplicate with three repetitions. Statistical analysis was carried out using the Statistical Package . One-way analysis of variance (ANOVA) was utilized to evaluate the statistical difference. All data were expressed as mean ± standard deviation. Comparison of means was carried out by Duncan's multiple range test significant difference  and WPM-Mb treated with DBD-CP  are shown in * value (negative) and remained stable level throughout the storage (p ≥ 0.05). The remaining groups (0–70 kV) exhibited the highest a* value at day 0. The a*-values at each storage point showed a decreasing trend with the processing voltage increased (0–70 kV). The decrease in redness from 5.32  to 3.63  demonstrated substantial changes in Mb during DBD-discharge at 70 kV, especially. For WPM-Mb, the decline in redness was related to the oxidation of Mb  to metMb, leading to the brownish-gray color  were notably lower than untreated (0 kV), illustrating that DBD-CP processing caused a rapid accumulation of metMb. The above experimental results indicated that (i) metMb, (ii) non-heme iron, (iii) intact heme released from globin, and (iv) ferryl-Mb/crosslink Mb were possible oxidative species that contributed to the DBD-CP-induced lipid oxidation. The results in The changes in the aay color . Moreovep ≥ 0.05). Obviously, non-heme content in the treated groups increased significantly (p < 0.05)  in 4 days. The results suggested that DBD-CP could markedly promote the release of non-heme iron. Furthermore, the denaturation of the porphyrin ring of Mb possibly occurred under cold plasma with high voltage conditions, causing the release of free iron called “non-heme iron.” In WPM-Mb, iron ions continued to accumulate during time. It has also been demonstrated that H2O2/O2O2 could facilitate heme degradation and release free iron in the gastric fluid model. Another study illustrated that H2O2, OH radicals, and UV radiation produced by CP treatment accelerated the heme degradation of horseradish peroxidase protein . Washing processes may remove water-soluble antioxidants, pro-oxidants, and some lipids from the muscle, thereby eliminating the interfering reactions caused by these factors (p < 0.05), while the 70 kV sample increased rapidly in the same period (p < 0.05), and followed by a stable trend by day 4 (p ≥ 0.05) (p < 0.05). The data showed that DBD-CP significantly enhanced PV in WPM-Mb (50-70 kV), indicating that DBD-CP promoted Mb-induced lipid oxidation in the washed muscle. MetMb could form ferryl-Mb to facilitate lipid oxidation  . In addixidation .p ≥ 0.05). For WPM-Mb (0 kV), a gradual increase (p ≥ 0.05) in TBARS was found during 0–3 days, followed by a notable increase after 3 days (p < 0.05). During the first 2 days, TBARS values in WPM-Mb (50–70 kV) were found to increase rapidly (p < 0.05), and no notable change was observed after that (p ≥ 0.05). During storage, WPM-Mb (50–70 kV) generally had higher TBARS values, compared with 0 kV samples (p < 0.05). Although the TBARS of all samples treated by DBD-CP increased with storage time, the rate of increase was higher for 70 kV samples than for 50–60 kV samples. Therefore, lipid oxidation catalyzed by Mb could be promoted via DBD-CP treatment, especially a higher voltage. What's more, it should be noted that PV and TBARS increased with increased voltage even without any storage of the washed muscle matrix containing Mb . This result suggested that free radical species and oxidants generated during 180 s of exposure caused considerable lipid oxidation. A possible good explanation was that radicals produced during DBD-CP processing could facilitate the generation of peroxides and lipid oxidation, increasing TBARS  at 0 to 4 days. The content of T-SH in WPM-Mb gradually decreased with the increase of voltages (50–70 kV) over the storage time (p < 0.05). The results demonstrated that DBD-CP could promote T-SH oxidation, and the higher the voltage intensity, the more the number of T-SH was oxidized. What's more, Feng et al. (2O2 and ROS). Thus, the loss of thiols in the washed pork muscle could be due to cysteine oxidation in the myofibrillar proteins and/or myofibrillar protein disulfide formation.Changes of T-SH contents in WPM are represented in g et al.  pointed SDS-PAGE was employed for characterizing the protein patterns of Mb. As depicted in p < 0.05), which also coincided with the changes in absorption spectra  with the treatment voltages at day 4. The increase of metMb ratio together with kox. The results suggested that the secondary structure of Mb was damaged by DBD-CP, which made the structure disordered. The disordered structure was enhanced with the treatment of 50 to 70 kV, in which the notable changes occurred in α-helix and random coil. According to reports, proteins could maintain their bioactivity through weak interactions such as hydrophobic, hydrogen, or ionic bonds  the conformation of Mb is altered by DBD-CP treatment; (ii) reactive species in DBD-CP promote Mb oxidation and lipid oxidation. Meanwhile, the changes of absorption spectra and autoxidation rate of Mb further confirm that reactive species produced by DBD-CP treatment promotes some release of heme from the globin, forming some apoMb. This process leads to lipid oxidation and the aggregation of some metMb and apoMb molecules, which affects the particle size distribution and the zeta potential. More importantly, with the increase of treatment voltages (0–70 kV), the structural change of Mb becomes more apparent. To sum up, DBD-CP accelerates the lipid oxidation in WPM-Mb mainly due to the reaction of reactive species in DBD-CP to promote the Mb oxidation and change the Mb conformation. Therefore, inhibition of lipid oxidation in pork or other muscle food systems containing heme proteins needs further studies to be performed in the future.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.XW and JW: data curation, writing-original draft, and data collection and analysis. ZW: data curation. WY, HZ, JZ, and JW: supervision and writing-review and editing. All authors have read and agreed to the published version of the manuscript.